1. Field of the Invention
The present invention relates to the field of in situ hybridization assays useful for detecting as few as 1-5 copies of target nucleic acid per cell. This assay method significantly increases the sensitivity of detection of nucleic acids over other known methods. In addition, this hybridization method is accomplished with far greater speed than has been reported for other in situ assays. This present invention also provides a method for the rapid and sensitive detection of nucleic acids and proteins in the same cell. A kit is provided for a simple one step fixation/hybridization in situ assay. 2. Description of the Prior Art
In situ hybridization provides a technique for the determination and quantitation of biopolymers such as nucleic acids (DNA and RNA) and proteins in tissues at the single cell level. Such hybridization techniques can detect the presence or absence of specific genes in tissues at the single cell level. In situ hybridization procedures may also be utilized to detect the expression of gene products at the single cell level.
By the use of specific nucleic acid (RNA or DNA) probes, genetic markers for infection and other disease states may be detected. Certain genetic diseases are characterized by the presence of genes which are not present in normal tissue. Other diseased conditions are characterized by the expression of RNAs or RNA translation products (i.e. peptides or proteins) which are not expressed in normal cells. Some disease states are characterized by the absence of certain genes or gene portions, or the absence or alteration of expression of gene products or proteins. Antibody probes specific for target antigenic biopolymers have also been used to identify the presence of viral proteins or gene products.
Current methods allow the detection of these markers but are relatively time consuming and of limited sensitivity. Hybridization techniques are based on the ability of single stranded DNA or RNA to pair (or hybridize) with a complementary nucleic acid strand. This hybridization reaction allows the development of specific probes that can identify the presence of specific genes (DNA), or polynucleotide sequences for the transcription and expression of those genes (mRNA).
Solution hybridization methods which require the destruction of the cell and the isolation of the nucleic acids from the cell prior to carrying out the hybridization reaction sacrifice the cellular integrity, spatial resolution and sensitivity of detection. In situ hybridization allows the detection of RNA or DNA sequences within individual cells. In situ hybridization yields greater sensitivity than solution hybridization by means of eliminating the dilution of a particular target gene, nucleic acid, or protein by the surrounding and extraneous RNA and DNA of other cells. In situ hybridization also allows for the simultaneous detection of multiple substances, i.e. genes, nucleic acids or proteins within individual cells, permitting the identification of a particular cell expressing a cellular gene or viral sequence. In addition, since in situ hybridization analysis is performed and quantitated for single cells, minimal sample and reagents are required.
Prior to the present invention, in situ hybridization procedures were only capable of detecting nucleic acids present at greater than ten copies per cell. Such procedures required multiple steps and at least 4 hrs. to over 14 days to perform.